Light source comprising a plurality of microstructured optical elements

ABSTRACT

The invention relates to a light source comprising a microstructured optical element ( 11 ) that receives and spectrally spreads the light from a primary light source ( 3 ). The inventive light source is characterised in that the spectrally spread light penetrates at least one other microstructured optical element ( 15, 19, 21 ). Said light source can be efficiently used in scanning microscopy and especially in STED microscopy.

The invention relates to a light source comprising a microstructuredoptical element that receives and spectrally spreads the light from aprimary light source.

Patent specification U.S. Pat. No. 6,097,870 discloses an arrangementfor generating a broadband spectrum in the visible and infrared spectralregions. The arrangement is based on a microstructured fiber into whichthe light from a pump laser is coupled. The pump light is spread in themicrostructured fiber by nonlinear effects. Use is made asmicrostructured fiber of so-called photonic band gap material or photoncrystal fibers, holey fibers or microstructured fibers. Configurationsas so-called hollow fibers are also known.

A further arrangement for generating a broadband spectrum is disclosedin the publication by Birks et al.: “Supercontinuum generation intapered fibers” Opt.Lett. Vol. 25, p. 1415 (2000). Use is made in thearrangement of a conventional optical fiber with a fiber core that has ataper at least along a segment. Optical fibers of this type are known asso-called tapered fibers.

Particularly in microscopy, endoscopy, flow cytometry, chromatographyand in lithography, universal illuminating devices of high luminance areimportant for illuminating the objects. In scanning microscopy, a sampleis scanned with a light beam. Lasers are frequently used as light sourcefor this purpose. An arrangement with a single laser emitting aplurality of laser lines, for example, is known from EP 0 495 930:

“Konfokales Mikroskopsystem für Mehrfarbenfluoreszenz” [“Confocalmicroscope system for polychromatic fluorescence”]. At present, it ismostly mixed gas lasers, in particular ArKr lasers, that are used.Biological tissues or sections prepared with the aid of fluorescentdyes, for example, are examined as a sample. In the field of materialexamination, the illuminating light reflected by the sample is oftendetected. Use is also frequently made of solid state lasers and dyelasers as well as of fiber lasers and optical parametric oscillators(OPOs) upstream of which a pump laser is arranged.

Laid-open patent application DE 101 15 488 A1 discloses an apparatus forilluminating an object that includes a microstructured optical elementthat spectrally spreads the light of a laser. The apparatus comprises anoptics that shapes the spectrally spread light to form an illuminatinglight beam. The laid-open patent application also discloses the use ofthe apparatus for illumination in a microscope, in particular in ascanning microscope.

Patent application DE 101 15 509 A1 discloses an arrangement forexamining microscopic preparations with the aid of a scanning microscopeand of an illuminating device for a scanning microscope. The arrangementcomprises a laser and an optical means that projects the light generatedby the laser onto a sample to be examined. Provided between the laserand the optical means is an optical component that spectrally spreads inthe light generated by the laser during a single pass, the opticalcomponent consisting of photonic band gap material and preferably beingdesigned as an optical fiber.

The generation of light of a broadband wavelength spectrum from 500 to1600 nm with the aid of an air/quartz-glass fiber is exhibited in thearticle by Ranka et al., Optics Letters, Vol. 25, No. 1.

Apart from depending on the wavelength of the primary light source, theproperties of the light generated with the aid of microstructuredoptical elements, such as photonic crystal fibers, for example, alsodepend on the parameters of the microstructured optical element such asfor example, the zero-dispersion wavelength or the type and thedimensions of the hole structure or microstructure. As a rule, given thesame wavelength of the primary light two different photonic crystalfibers have a different emission spectrum. This is particularlydisadvantageous, in particular, with regard to the reproducibility ofexperiments.

As a rule, the power of the spectrally spread light is distributed in alargely uniform fashion over the entire broad spectral region such thatonly a relatively slight light power (typically 1-5 mW/nm) is availablefor applications in which only light of individual wavelengths or lightof an individual small wavelength region is required.

Laid-open patent application DE 100 56 382 A1 discloses a light sourcefor illuminating in scanning microscopy and a scanning microscope. Thelight source contains an electromagnetic energy source that emits lightof one wavelength, and a means for spatially dividing the light into atleast two partial light beams. An intermediate element for changingwavelength is provided in at least one partial light beam. The lightsource can be used in STED microscopy.

In scanning microscopy, a sample is illuminated with a light beam inorder to observe the reflected light or fluorescent light emitted by thesample. The focus of an illuminating light beam is moved in an objectplane with the aid of a controllable beam deflecting device, generallyby tilting two mirrors, the deflection axes mostly being perpendicularto one another such that one mirror deflects in the x-direction and, andthe other in the y-direction. The tilting of the mirrors isaccomplished, for example, with the aid of galvanometer actuatingelements. The power of the light coming from the object via a detectionbeam path is measured with the aid of a detector as a function of theposition of the scanning beam. The actuating elements are usuallyequipped with sensors for determining the current mirror position.

An object is scanned in three dimensions with the focus of a light beamspecifically in confocal scanning microscopy.

A confocal scanning microscope generally comprises a light source, animaging optics with the aid of which the light from the source isfocused onto a pinhole diaphragm—the so-called excitation diaphragm—abeam splitter, a beam deflecting device for beam control, a microscopeoptics, a detection stop and the detectors for detecting the detectionlight or fluorescent light. The illuminating light is often coupled invia the beam splitter, which can, for example, be designed as a neutralbeam splitter or as a dichroic beam splitter. Neutral beam splittershave the disadvantage that a great deal of excitation light or a greatdeal of detection light is lost depending on the splitting ratio.

The detection light (for example fluorescent light or reflection light)coming from the object passes back via the beam deflecting device to thebeam splitter, passes the latter and is subsequently focused onto thedetection stop behind which the detectors are located. Detection lightthat does not originate directly from the focus region takes a differentlight path and does not pass the detection stop, and so punctiforminformation is obtained that leads by means of sequential scanning ofthe object to a three-dimensional image. A three-dimensional image ismostly obtained by layerwise image data acquisition, the track of thescanning light beam ideally describing a meandering line on and/or inthe object (scanning a line in the x-direction for a constanty-position, subsequently stopping x-scanning and pivoting byy-adjustment to the next line to be scanned and then, for a constanty-position, scanning this line in a negative x-position, etc). In orderto enable layerwise image data acquisition, the sample stage or theobjective is displaced after the scanning of a layer, and in this waythe next layer to be scanned is brought into the focal plane of theobjective.

In many applications, samples are prepared with a plurality of markers,for example a plurality of different fluorescent dyes. These dyes can beexcited sequentially, for example with the aid of illuminating lightbeams that have different excitation wavelengths.

As is described in European patent EP 0 491 289 entitled“Doppelkonfokales Rastermikroskop” [“Double confocal scanningmicroscope”], it is possible to achieve an increase in resolution in thedirection of the optical axis by means of a double objective arrangement(4Pi arrangement). The light coming from the illuminating system issplit into two partial beams that illuminate the sample simultaneouslyin a fashion running opposite to one another through two objectivesarranged with mirror symmetry. The two objectives are arranged ondifferent sides of the object plane common to them. This interferometricillumination forms an interference pattern at the object point thatexhibits a primary maximum and a plurality of secondary maxima inconjunction with constructive interference. By comparison with theconventional scanning microscope, owing to the interferometricillumination an increased axial resolution can be achieved with the aidof a double confocal scanning microscope.

An arrangement for raising the resolving power for fluorescenceapplications is known from DE 44 16 558. Here, the lateral edge regionsof the focus volume of the exciting light beam are illuminated with theaid of a light beam of another wavelength, the so-called stimulationlight beam, which is emitted by a second laser in order to bring thesample regions excited by the light from the first laser back into theground state in a stimulated fashion. It is now only the spontaneouslyemitted light from the regions not illuminated by the second laser thatis detected, and so an overall improvement in resolution is achieved.The designation of STED (Stimulated Emission Depletion) has been adoptedfor this method.

A new development has shown that it is possible to achieve animprovement in resolution simultaneously both laterally and axially whensuccess is achieved in hollowing out the focus of the stimulation lightbeam. To this end, there is introduced into the beam path of thestimulation light beam a round phase delay plate that delays the lightwaves in subregions by a phase that corresponds to an optical pathlength of λ/2. The phase delay plate has a diameter smaller than thebeam diameter and is therefore overilluminated. In order to achieve astimulation beam hollow in the inside, the light quantity thatexperiences a phase delay of λ/2 must be equal to the light quantity notdelayed.

STED microscopy is currently being carried out in three differentconfigurations:

By means of a titanium-sapphire (TiSa) laser for stimulateddeenergization of the fluorescent dye, and of an optical parametricoscillator (OPO), pumped by the TiSa, for exciting the fluorescent dye(Proc. Natl. Acad. Sci. U.S.A., Vol. 97, p. 8206-8210, 2000).

By means of two synchronized laser diodes of which one laser diode has awavelength in the wavelength region of the absorption spectrum of thedye, and the other laser diode has a wavelength in the region of theemission spectrum of the dye (Appl. Phys. Lett., Vol. 82, No. 18, p.3125-3127, 2003).

By means of a pulsed solid state laser whose light is used, on the onehand, for stimulated deenergization of the fluorescent dye. On the otherhand, the light is doubled in frequency and used to excite the dye.(Hell, S.W. (1997). “Increasing the Resolution of Far-Field FluorescenceMicroscopy by Point-Spread-Function Engineering.” Topics In FluorescenceSpectroscopy 5: Nonlinear and Two-Photon-Induced Fluorescence. J.Lakowicz. New York, Plenum Press. 5.)

Titanium-sapphire lasers, for example, are used as light source in STEDmicroscopy in conjunction with the optical parametric oscillators(OPOs). Light sources of this type have the disadvantage that they canmake available only light of a very limited wavelength spectrum andthat, moreover, they are difficult to operate. The very high procurementprice is not the least of the disadvantages with these light sources.Mutually synchronized semiconductor lasers are also currently being usedas light sources in STED microscopy, the light power of the laser diodeused for stimulated deenergization often disadvantageously notsufficing. Moreover, operation is necessarily restricted to twowavelengths of the laser diodes used. As an alternative, solid statelasers with subsequent frequency doubling are also currently being usedin STED microscopy. Two mutually independent wavelengths are herebynecessarily fixed for the light for exciting the sample, and for thelight that effects a stimulated emission, and this limits the ability touse this type of light source to a few possible applications.

It is the object of the present invention to specify a light sourcecomprising a microstructured optical element whose emission spectrum isadapted to the respective application, and that can be used, inparticular, in scanning microscopy and, specifically, in STEDmicroscopy.

The object is achieved by means of a light source characterized in thatthe spectrally spread light traverses at least one furthermicrostructured optical element.

Owing to the sequential arrangement of two or more microstructuredoptical elements, the spectral properties of the light emitted by thelight source can be influenced and adapted to the requirements of theintended application. In particular, the power of the light emitted bythe light source can be increased in the spectral subregions that are ofparticular importance for the application by a suitable selection of theparameters of the microstructured optical element and of the furthermicrostructured optical element. For example, when the light source isused in STED microscopy it is possible to maximize the light power inthe region of the absorption spectrum of the sample dyes used and in theregion of the emission spectrum of the sample dyes used. The inventivelight source is therefore particularly suitable for applications inhigh-resolution microscopy such as, for example, the abovementioned STEDmicroscopy or in STED-4Pi scanning microscopy (double confocal scanningmicroscope), and in CARS microscopy.

The inventive light source can advantageously be used to generateemission light whose spectral width exceeds the spectral width that eachindividual microstructured optical element would generate. Such a lightsource is of interest, in particular, for multiwavelength STEDapplications, since a very wide supercontinuum is required here.

In a very particularly preferred variant refinement of the light source,the microstructured optical element and the further microstructuredoptical element are spliced together. The splicing of optical fibers isa technique adequately known to the person skilled in the art. Inlaid-open patent application US 2003/0081915 it is described, inaddition, how a conventional fiber and a microstructured fiber can bespliced together such that the transmission losses are minimized.

In another preferred variant refinement of the light source, the lightthat emerges from the microstructured optical element is coupled intothe further microstructured optical element by a lens arrangement.

Pump-probe experiments can also be carried out efficiently with the aidof the inventive light source.

The primary light source is preferably a pulsed light source, andcomprises in a preferred variant a pulsed laser that can be designed,for example, as a pulsed titanium sapphire laser.

In a particularly preferred embodiment, a means is provided forselecting light components over at least one wavelength and/or at leastone wavelength region. These means can be, for example, color filters ordichroic filters. The means for selection preferably include anacousto-optical or electro-optical component. In a preferred variant,the means for selection is designed as an AOTF (Acousto Optical TunableFilter) or as an AOBS (Acousto Optical Beam Splitter).

As already mentioned, the light source is also eminently suitable withina method for generating illuminating light for STED microscopy or forpump-probe experiments. A light component that has a wavelength withinthe excitation spectrum of the respectively used fluorescence dye ishereby split off from the spectrally spread light, emitted by the lightsource, with the aid of the means for selecting a light component and afurther light component, which has a wavelength within the emissionspectrum of the fluorescence dye used, is split up and formed into anilluminating light beam. Whereas the light component that has awavelength within the excitation spectrum of the fluorescence dye servesto excite the sample in the illuminated region, the light component thathas a wavelength within the emission spectrum serves for triggeringstimulated emission in a sample region partially overlapping theexcitation sample region. When the primary light source is a pulsedlight source, the pulses in the two split off light components arenecessarily mutually synchronized, which is a very important propertyfor STED microscopy.

The light from the primary light source preferably traverses themicrostructured optical element and/or the further microstructuredoptical element only once. However, a repeated traversal is alsopossible.

The microstructured optical element and/or the further microstructuredoptical element preferably contains photonic band gap material. Themicrostructured optical element and/or the further microstructuredoptical element are/is preferably designed as optical fiber(s) (photoniccrystal fiber (PCS); holey fiber, etc).

In another variant, the microstructured optical element configured as anoptical fiber has a taper (tapered fiber).

In a preferred embodiment of the scanning microscope, themicrostructured optical element and/or the further microstructuredoptical element is assembled from a multiplicity of micro-opticalstructural elements that have at least two different optical densities.A very particular preference is for a refinement in which the opticalelement includes a first region and a second region, the first regionhaving a homogeneous structure, and a microscopic structure composed ofmicro-optical structural elements is formed in the second region. It isalso advantageous when the second region surrounds the first region. Themicro-optical structural elements are preferably cannulars, webs,honeycombs, tubes or cavities.

In a particular variant, the microstructured optical element and/or thefurther microstructured optical element comprise/comprises glass orplastic material arranged next to one another and cavities. A particularpreference is the variant design in which the microstructured opticalelement and/or the further microstructured optical elementcomprise/comprises photonic band gap material and is configured as anoptical fiber. It is preferred to provide between the laser and theoptical fiber an optical diode that suppresses backreflections of thelight beam that originates from the ends of the optical fiber.

A design variant that is of very particular preference and easy toimplement includes as microstructured optical element and/or as furthermicrostructured optical element a conventional optical fiber with afiber core diameter of approximately 9 μm that has a taper at leastalong a segment. Optical fibers of this type are known as so-calledtapered fibers. The optical fiber is preferably 1 m long overall and hasa taper of a length of 30 mm to 90 mm. In a preferred refinement, thediameter of the entire fiber is approximately 2 μm in the region of thetaper.

A further preferred variant embodiment includes a microstructuredoptical element and a further microstructured optical element in thecase of which elements the structural elements merge into one anothercontinuously. In a very particularly preferred variant, amicrostructured optical element and a further microstructured opticalelement are designed as optical fibers with a continuous transition.

The inventive light source can also, for example, be used in a flowcytometer or an endoscope or a chromatograph or a lithography apparatus.

The subject matter of the invention is illustrated schematically in thedrawing and described below with the aid of the figures, in which:

FIG. 1 shows an inventive light source,

FIG. 2 shows a further inventive light source, and

FIG. 3 shows an inventive confocal scanning microscope.

FIG. 1 shows an inventive light source 1 having a primary light source 3that is configured as a pulsed titanium sapphire laser 5. The light 7from the primary light source is coupled with the aid of the incouplingoptics 9 into a microstructured optical element 11 that is designed as aphotonic crystal fiber 13. Spliced directly to the photonic crystalfiber 13 is a further microstructured optical element 15 that isdesigned as a further photonic crystal fiber 17. Following similarly athird and a fourth microstructured optical element 19, 21 are spliced tothe third and fourth photonic crystal fibers 23, 25. The spectrallyspread light emerging from the fourth photonic crystal fiber 25 isshaped into an illuminating light beam 29 with the aid of the optics 27.The illuminating light beam 29 subsequently traverses a means 31 forselecting light components of at least one wavelength and/or at leastone wavelength region, which is designed as an AOTF 33. The illuminatinglight beam 29 emerging from the AOTF 33 now includes only lightcomponents of the selected wavelength or of the selected wavelengthregions, while the remaining light components are directed by the AOTFinto a beam trap (not shown) . The light source has a housing 35 for thepurpose of protection against external influences, in particularprotection against contamination.

A further inventive light source is illustrated in FIG. 2. The light 7from the primary light source 3 is firstly coupled into a conventionaloptical fiber 12 with the aid of the incoupling optics 9. Theconventional optical fiber 12 is spliced to a microstructured opticalelement 11 that is designed as a photonic crystal fiber 13. The light 7is spectrally spread in the photonic crystal fiber 13 and coupled out ofthe fiber. The spectrally spread light 16 is subsequently coupled withthe aid of a lens arrangement 14 into a further microstructured opticalelement 15, which is equipped as further photonic crystal fiber 17. Thecoupling of two optical fibers to a lens arrangement is standard infiber optics and can be ready made. Located following the furtherphotonic crystal fiber 17 is a third microstructured element 19comprising a third photonic crystal fiber 23. In the transition region20, which is illustrated with a gradual gray transition, the structuralelements merge into one another continuously. After traversing all theoptical elements, the light beam has a spectrum in which a particularlylarge quantity of light has been converted into specific spectralregions by comparison with all the other spectral regions. Thisspectrally shaped light beam 28 subsequently traverses a means 31 forselecting light components of at least one wavelength and/or at leastone wavelength region, which is designed as an AOTF 33. Subsequently,the spectrally shaped light beam 28 is split with the aid of a beamsplitter 36 into an excitation light beam 30 and a stimulation lightbeam 32. The stimulation light beam 32 traverses a phase delay plate 34such as is used in STED microscopy. This mode of procedure is adequatelyknown to the person skilled in the art. The two light beams are reunitedwith one another via a beam recombiner 38. As described in FIG. 3, thislight beam can subsequently be coupled as illuminating light beam 29into an inventive scanning microscope and used for the purpose of STEDmicroscopy.

FIG. 3 shows an inventive scanning microscope that is designed as aconfocal scanning microscope. The illuminating light beam 29 emanatingfrom an inventive light source 1 having the microstructured opticalelements (not shown in this figure) is focused by the lens 61 onto theilluminating pinhole diaphragm 37 and passes subsequently to the mainbeam splitter 39, which directs the illuminating light beam 29 to thebeam deflecting device 41, which includes a cardanically suspendedscanning mirror 43. The beam deflecting device 41 guides theilluminating light beam 29 through the scanning lens 45 and the tubelens 47 as well as through the objective 49 via or through the sample51. Detection light 53 emanating from the sample and which isillustrated by dashes in the figure passes back, on the reversed lightpath, specifically through the objective 49, the tube lens 47 andthrough the scanning lens 45, to the beam deflecting device 41 and tothe main beam splitter 39, passes the latter and, after traversing thedetection pinhole diaphragm 55, passes to the detector 57 that isdesigned as a multiband detector 59. The detection light is detected invarious spectral detection channels in the multiband detector 59, andgenerates electrical signals proportional to the power that are relayedto a processing system (not shown) for displaying an image of the sample51.

The invention has been described with reference to a particularembodiment. However, it goes without saying that changes andmodifications can be carried out without departing in so doing from thescope of protection of the following claims.

LIST OF REFERENCE NUMERALS

-   1 light source-   3 primary light source-   5 titanium sapphire laser-   7 light-   9 incoupling optics-   11 microstructured optical element-   12 conventional optical fiber-   13 photonic crystal fiber-   14 lens arrangement-   15 further microstructured optical element-   16 spectrally spread light-   17 further photonic crystal fiber-   19 third microstructured optical element-   20 transition region-   21 fourth microstructured optical element-   23 third photonic crystal fiber-   25 fourth photonic crystal fiber-   27 optics-   28 spectrally shaped light beam-   29 illuminating light beam-   30 excitation light beam-   31 mean for selecting light components-   32 stimulation light beam-   33 AOTF-   34 phase delay plate-   35 housing-   36 beam splitter-   37 illuminating pinhole diaphragm-   38 beam recombiner-   39 main beam splitter-   41 beam deflecting device-   43 scanning mirror-   45 scanning lens-   47 tube lens-   49 objective-   51 sample-   53 detection light-   55 detection pinhole diaphragm-   57 detector-   59 multiband detector-   61 lens

1. A light source comprising a microstructured optical element thatreceives and spectrally spreads the light from a primary light source,characterized in that the spectrally spread light traverses at least onefurther microstructured optical element.
 2. The light source as claimedin claim 1, characterized in that the microstructured optical elementand/or the further microstructured optical element contains photonicband gap material.
 3. The light source as claimed in claim 1,characterized in that the microstructured optical element and/or thefurther microstructured optical element are/is designed as opticalfiber(s).
 4. The light source as claimed in claim 3, characterized inthat the microstructured optical element and/or the furthermicrostructured optical element have/has a taper (tapered fiber).
 5. Thelight source as claimed in claim 3, characterized in that themicrostructured optical element and the further microstructured opticalelement merge into one another continuously.
 6. The light source asclaimed in claim 1, characterized in that the microstructured opticalelement and/or the further microstructured optical element are/is aphotonic crystal fiber (microstructured fiber, holey fiber).
 7. Thelight source as claimed in claim 1, characterized in that themicrostructured optical element and the further microstructured opticalelement are spliced together.
 8. The light source as claimed in claim 1,characterized in that the light that emerges from the microstructuredoptical element can be coupled into the further microstructured opticalelement with the aid of a lens arrangement.
 9. The light source asclaimed in claim 1, characterized in that the primary light sourcecomprises a pulsed laser.
 10. The light source as claimed in claim 1,characterized in that the light from the primary light source repeatedlytraverses the microstructured optical element and/or the furthermicrostructured optical element.
 11. The light source as claimed in 9claim 1, characterized in that means are provided for selecting lightcomponents over at least one wavelength and/or at least one wavelengthregion.
 12. The light source as claimed in claim 1, characterized by usein a flow cytometer or an endoscope or a chromatograph or a lithographyapparatus.
 13. A microscope having a light source as claimed in claim 1.14. A scanning microscope having a light source as claimed in claim 1.15. The scanning microscope as claimed in claim 14, characterized inthat the scanning microscope is a confocal scanning microscope and/or adouble confocal scanning microscope and/or an STED scanning microscopeand/or an STED-4Pi scanning microscope and/or a CARS scanningmicroscope.
 16. A method for generating illuminating light,characterized by the following steps: generating spectrally spread lightwith the aid of a light source as claimed in claim 1, selecting at leastone illuminating light wavelength and/or at least one illuminating lightwavelength region, and splitting off the illuminating light of the atleast one illuminating light wavelength and/or of the at least oneilluminating light wavelength region from the spectrally spread light.17. The method as claimed in claim 16, characterized in that theilluminating light optically excites a sample.
 18. The method as claimedin claim 16, characterized by the further step of: selecting at leastone further illuminating light wavelength and/or at least one furtherilluminating light wavelength region, and splitting off furtherilluminating light of the at least one further illuminating lightwavelength and/or of the at least one further illuminating lightwavelength region from the spectrally spread light.
 19. The method asclaimed in claim 18, characterized in that the further illuminatinglight effects a stimulated emission.
 20. The use of the method asclaimed in claim 16 in STED microscopy.
 21. The use of the method asclaimed in claim 16 for carrying out pump-probe experiments.